Vesicular release of the biogenic amine neurotransmitters serotonin (5-hydroxytryptamine, 5HT), norepinephrine (NE), and dopamine (DA) is followed by the rapid clearance of each molecule through specialized presynaptic transport proteins. The control of synaptic signaling achieved by neurotransmitter uptake is most clearly evident from the addictive and therapeutic activities of transporter inhibitors, including cocaine and antidepressants, which markedly elevate extracellular 5HT, NE, and DA levels, affect neuronal firing rates, and alter behavior. Due to an absence of cloned transporter genes, molecular details of monoamine transporter structure, function, and regulation have been largely unavailable. Over the past two years, we have achieved the first cloning of rodent and human 5HT transporter (SERT) cDNAs and characterized these clones in transient and stable nonneuronal cell hosts using radioligand uptake and binding paradigms as well as single cell biophysical approaches. We have developed SERT antibodies suitable for transporter immunoprecipitation, immunoblotting, and immunocytochemistry, demonstrated second messenger-based regulation of SERT mRNA levels, and identified human genomic clones likely to contain SERT transcription control elements. In-the present proposal, we seek to build upon these efforts to delineate critical structural and regulatory elements for SERT expression. Specifically, we will 1) use site-directed mutagenesis and chimeric SERTs to delineate important domains and residues on SERTs required for substrate (ion, 5HT) and antagonist (cocaine, antidepressant) recognition, 2) use SERT antibodies to visualize SERTs in the rodent brain and to determine important constitutive and regulated posttranslational modifications (glycosylation, phosphorylation) of SERT proteins, 3) use whole cell patch clamp techniques to reveal the properties of SERTs as a function of membrane potential in single cells, and 4) use model cell systems and identified SERT genomic clones to elucidate the structural basis for constitutive and regulated SERT gene expression.